This invention relates to agents that may be used to regulate mitosis in eucaryotic cells. Specifically, novel human BUB genes and their encoded proteins are disclosed. These genes and proteins may be used as targets for the development of novel agents that inhibit aberrant cellular proliferation in tumor cells.
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
The production of two viable and equivalent daughter cells in mitosis requires that events leading to cell division proceed in a carefully ordered fashion. Among other tightly regulated events, replicated sister chromosomes must be properly segregated, one to each daughter cell. For this reason, mitosis cannot be allowed to proceed if the genome has not been fully replicated or if chromosomes are not properly attached to a fully assembled mitotic spindle. Mechanisms required for ensuring the dependency of cell division on completion of such prerequisite steps have been termed mitotic checkpoints (52).
This checkpoint mechanism directly monitors the spatial position of chromosomes within the spindle and applies this information to regulate the activities of proteins that induce chromosome separation and allow the cell to exit mitosis and complete cell division (2, 3, 4). The mitotic checkpoint has sufficient sensitivity to detect the presence of a single unaligned chromosome amidst tens of chromosomes that are aligned (5). Furthermore, this checkpoint will block the cell in mitosis by delaying the onset of anaphase for many hours so that the unaligned chromosome has ample opportunity to establish the spindle connections that allow alignment at the spindle equator. Studies in yeast, insect cells and vertebrate cells show that this mitotic checkpoint monitors the kinetochore as a means to determine whether chromosomes have achieved metaphase alignment. As the kinetochore is the chromosomal site for microtubule attachment and contains molecular motors that specify various aspects of chromosome movement (6, 7), the checkpoint is likely to recognize biochemical signals at the kinetochore that differ between unaligned and aligned chromosomes (4). Consistent with this possibility, laser ablation of a single unattached kinetochore of a monopolar chromosome will abrogate the checkpoint arrest and the chromosomes aligned at the equator will separate as the cell enters anaphase (8). These experiments also demonstrate that checkpoint delay is mediated by an inhibitory signal from the unattached kinetochore. Interestingly, there is a reproducible lag of approximately 20 minutes from the time that the last chromosome becomes aligned till the onset of anaphase (8). This lag may define the time required to complete the biochemical reactions that are necessary for turning off the checkpoint induced block and the activation of the cyclosome/APC complex that specifies anaphase onset through ubiquitin-mediated proteolysis (9). To date, the nature of the signal that the kinetochore emits when it is not properly aligned has not yet been elucidated.
Kinetochores generate motive force through interactions with microtubules. For a bipolar attached chromosome, the tendency for each of the two oppositely-faced kinetochores to move towards their respective poles generates tension between the kinetochore pair. This contrasts with an unattached or a monopolar chromosome whereby little or no tension is generated at the kinetochores.
Elegant micromanipulation experiments performed on the trivalent sex chromosomes in the mantis spermatocyte (10) suggested that kinetochore tension serves as the signal which is detected by the mitotic checkpoint apparatus. During meiosis I, the two X chromosomes pair with a single Y chromosome. On occasion, one of the X""s fails to pair and its mono-orientation delays anaphase. If kinetochore tension was supplied by using a microneedle to pull the unattached kinetochore in the direction of the opposite pole, the block to anaphase was lifted and all the chromosomes that were at the spindle equator separated and moved poleward. Again, there was a reproducible time-lag of approximately 15 minutes between application of kinetochore tension on the unaligned X chromosome and separation of the chromosomes aligned at the equator.
At a biochemical level, the inhibitory signal emitted from the unattached kinetochore is likely due to the specific phosphorylation of kinetochore proteins whose identities remain unknown. This hypothesis arises from the observation that a monoclonal antibody 3F3/2 (11), specific for undefined phosphoproteins, recognized differentially expressed phosphoepitopes at kinetochores in PtK cells (12). 3F3/2 was isolated from antibodies generated against mitotic frog extracts that had been incubated with ATP-xcex3S so that proteins would be selectively thio-phosphorylated by mitotic kinases (11). Although the precise identity of the 3F3/2 epitope is unknown, it recognizes a small subset of undefined proteins that are phosphorylated in mitosis. It is unknown whether proteins that contain the 3F3/2 epitope overlap with the more familiar MPM-2 phosphoepitope that also appears primarily in mitosis (13). The connection between 3F3/2 phosphorylation and the mitotic checkpoint was based on the early observation that 3F3/2 staining was invariably more prominent at the kinetochore of unaligned chromosomes in PtK cells. In contrast, staining was lost or greatly diminished at the kinetochores of chromosomes that were aligned at the spindle equator. If mAb 3F3/2 was microinjected into PtK cells, the kinetochore-bound antibodies did not interfere with chromosome alignment but they failed to separate even though they were aligned at the spindle equator (14). Thus, the 3F3/2 phosphoepitope(s) at the kinetochore are probably not involved with kinetochore motility. Consistent with the idea that unaligned kinetochores emit a signal that blocks the onset of anaphase, the persistence of the 3F3/2 phosphorylated proteins at the kinetochore of aligned chromosomes, as a result of their association with the injected 3F3/2 antibody, was believed to maintain the checkpoint delay.
An important advance towards resolving the biochemical mechanism by which the checkpoint detects kinetochore tension was made by showing that tension regulated 3F3/2 phosphorylation (18). In a series of micromanipulation experiments using grasshopper spermatocytes, it was shown that when a chromosome was experimentally displaced from the metaphase plate, the intensity of 3F3/2 staining was increased over the levels found at aligned kinetochores. The same observation was made in unmanipulated cells, where the absence of tension resulting from the erroneous attachment of both kinetochores to the same pole produced intense 3F3/2 staining at kinetochores. Importantly, when tension was experimentally introduced by pulling one of the kinetochores of the maloriented chromosome, 3F3/2 staining was lost at the kinetochore under tension while bright staining was still detected at the sister kinetochore that was not under tension. The combined data strongly suggest that the phosphorylation states of kinetochore proteins are regulated by the amount of tension that is exerted at kinetochores. The issue of how 3F3/2 phosphoproteins (and other b)biochemical changes) at the kinetochore are detected is unknown.
As discussed above, during cell division, quality control mechanisms monitor critical events, such as DNA replication, cell growth, and chromosome segregation. In most cases, these checkpoint systems will override the underlying cell-cycle machinery and block advancement into the next stage of the cell cycle until certain requirements are met in the current cell cycle stage. Although key regulators of mitotic progression such as cyclinB/cdc2kinase and the cyclosome/Anaphase Promoting Complex (APC) have been identified and characterized, their relationships with mitotic checkpoint control remains unclear.
The present inventors have appreciated a need for the elucidation of the essential components involved in the regulation of cellular proliferation will provide novel targets for arresting mitosis in transformed cells. Such targets may be used to advantage to identify novel anti-proliferative agents.
This invention provides novel, biological molecules useful for identification, detection, and/or molecular characterization of components involved in the regulation of mitosis. According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes an isolated open reading frame encoding a kinase of a size between about 115 and 130 kilodaltons. The encoded protein, referred to herein as huBUB1A, comprises a tripartite domain structure including an amino terminal kinetochore targeting domain, a central xcex1-helical coil domain and a carboxy terminal kinase domain. The protein demonstrates significant binding affinity for CENP-E, a kinesin-related motor protein which localizes to the kinetochore where it functions to promote alignment of chromosomes at the spindle equator. HuBUB1A is a cytoplasmic protein during interphase but is assembled onto kinetochores prior to CENP-E sometime in prophase.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human BUB1A protein. In a particularly preferred embodiment, the human BUB1A protein has an amino acid sequence the same as Sequence I.D. No. 2. An exemplary huBUB1A nucleic acid molecule of the invention comprises Sequence I.D. No. 1.
According to another aspect of the invention, a second isolated nucleic acid molecule is provided which includes an isolated nucleic acid encoding a kinase of a size between about 110 and 140 kilodaltons and appears to be the human homolog of mouse BUB1. The encoded protein, referred to herein as huBUB1B, demonstrates significant binding affinity for CENP-F, a protein involved in the assembly and formation of a mature trilaminar kinetochore. HuBUB1B protein is detectable in nuclei of cells that are in the G2 stage of interphase. The HuBUB1B protein is subsequently localized it kinetochores sometime in prophase, prior in time to huBUB1A assembly at the kinetochore.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human BUB1B protein. In a particularly preferred embodiment, the human BUB1B protein has an amino acid sequence the same as Sequence I.D. No. 4. An exemplary huBUB1B nucleic acid molecule of the invention comprises Sequence I.D. No. 3.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which includes an isolated open reading frame encoding a protein of a size between about 35 and 40 kilodaltons. The encoded protein, referred to herein as huBUB3, comprises five WD-40 motif repeats and complexes with huBUB1A and huBUB1B. The huBUB3 protein is a nuclear protein during interphase but also localizes to the kinetochores during mitosis.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a nucleic acid sequence encoding a human BUB3 protein. In a particularly preferred embodiment, the human BUB3 protein has an amino acid sequence the same as Sequence I.D. No. 6. An exemplary huBUB3 nucleic acid molecule of the invention comprises Sequence I.D. No. 5.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) Sequence I.D. No. 1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 1; (3) a sequence encoding preselected portions of Sequence I.D. No. 1, (4) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 2; (5) Sequence I.D. No. 3; (6) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 3; (7) a sequence encoding preselected portions of Sequence I.D. No. 3, (8) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 4; (9) Sequence I.D. No. 5; (10) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 5; (11) a sequence encoding preselected portions of Sequence I.D. No. 5, (12) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 6. Such partial sequences are useful as probes to identify and isolate homologues of the BUB genes of the invention. Accordingly, isolated nucleic acid sequences encoding natural allelic variants of the nucleic acids of Sequence I.D. Nos., 1, 3 and 5 are also contemplated to be within the scope of the present invention. The term natural allelic variants will be defined hereinbelow.
According to another aspect of the present invention, isolated human BUB1A, BUB1B and BUB3 proteins are provided. HuBUB1A is a protein kinase with a deduced molecular weight of between about 115 kDa and 130 kDa. HUBUB1A comprises a tripartite domain structure including an amino terminal kinetochore targeting domain, a central xcex1-helical coil domain and a carboxy terminal kinase domain. The protein demonstrates significant binding affinity for CENP-E, a kinesin-related protein which localizes to the kinetochore. Human BUB1B protein is between about 110 and 140 kilodaltons and appears to be the human homolog of mouse BUB1. The encoded protein is a protein kinase and demonstrates significant binding affinity for CENP-F, a protein involved in the assembly and formation of a mature trilaminar kinetochore. HuBUB3 protein is between about 35 and 40 kilodaltons in size. HuBUB3 comprises five WD-40 motif repeats and complexes with huBUB1A. The huBUB3 protein also localizes to the kinetochores during mitosis.
In preferred embodiments of the invention, the proteins are of human origin, and have the amino acid sequences the same as Sequence I.D. Nos. 2, 4 and 6 respectively. In a further embodiment the proteins may be encoded by natural allelic variants of Sequence I.D. Nos. 1, 3, or 5.Inasmuch as certain amino acid variations may be present in human BUB proteins encoded by a natural allelic variants, such proteins are also contemplated to be within the scope of the invention.
According to another aspect of the present invention, antibodies immunologically specific for each of the three different human BUB proteins described hereinabove are provided.
In yet another embodiment of the invention, methods are provided for identifying novel therapeutic reagents for the regulation of mitosis. The method entails the use of nucleic acid fragments for expressing predetermined domains of the BUB proteins of the invention. Exemplary domains include the kinase domains of huBUB1A, and huBUB1B or the kinetochore binding domains of any of the three BUB proteins. Exemplary domains for huBUB1A, for example, include but are not limited to 1) the domain involved in huBUB3 binding between amino acids 194-466; 2) the domain that binds CENP-E between amino acids 408-546; 3) the domain by which huBUB1A binds the kinetochore between amino acids 1-466; and 4) the kinase domain of the protein between amino acids 644- to the carboxy terminus. The peptide domains are expressed, purified, and then immobilized on a solid support. The immobilized peptides are then exposed to test compounds. Compounds which bind the immobilized peptides will be further characterized in in vitro kinase assays and immunoprecipitation assays. Those compounds which demonstrate inhibition of kinase activity or disruption of BUB/kinetochore assembly, will then be assessed for inhibition of cell growth in tumor cells.
In an alternative embodiment, the test compounds may be immobilized to the solid support, and then exposed to the BUB proteins of the invention.
In yet another embodiment, full length BUB proteins may be utilized in the methods described above.
The methods of the invention also comprise the use of recombinant cell lines expressing one or a subset of the BUB genes of the invention. As above, test compounds will be added to the recombinant cells or lysates prepared therefrom, and disruption of BUB/kinetochore complex binding and/or inhibition of kinase activity will be assessed.
Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specification and claims. The terms xe2x80x9cspecifically hybridizing,xe2x80x9d xe2x80x9cpercent similarityxe2x80x9d and xe2x80x9cpercent identity (identical)xe2x80x9d are defined in detail in the description set forth below.
With reference to nucleic acids of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used.
This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5xe2x80x2 and 3xe2x80x2 directions) in the naturally occurring genome of the organism from which it originates. For example, the xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
With respect to RNA molecules of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a xe2x80x9csubstantially purexe2x80x9d form (the term xe2x80x9csubstantially purexe2x80x9d is defined below).
With respect to protein, the term xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in xe2x80x9csubstantially purexe2x80x9d form.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to antibodies of the invention, the term xe2x80x9cimmunologically specificxe2x80x9d refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., huBUB1A, huBUB1B or huBUB3), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to nucleic acids and oligonucleotides, the term xe2x80x9cspecifically hybridizingxe2x80x9d refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). When used in reference to a double stranded nucleic acid, this term is intended to signify that the double stranded nucleic acid has been subjected to denaturing conditions, as is well known to those of skill in the art. In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The nucleic acids, proteins and antibodies of the present invention may be used to advantage as targets for the development of novel agents that inhibit aberrant cellular proliferation in tumor cells.
The human BUB molecules described above may also be used as research tools and will facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the regulation of cell division and mitotic checkpoint control.